Mucin biosynthesis

Purification and characterization of a mucin β6N-acetylglucosaminyltransferase

P. A. Ropp, M. R. Little, Pi-Wan Cheng

Research output: Contribution to journalArticle

66 Citations (Scopus)

Abstract

We have purified, to apparent homogeneity, a mucin β6N-acetylglucosaminyltransferase (β6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Galβ1-3GalNAcαbenzyl (Bzl) to stabilize the β6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Super-flow affinity column equilibrated with 1 mM Galβ1-3GalNAcαBzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 μmol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the β6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The K(m) values for UDP-GlcNAc and Galβ1-3GalNAcαBzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified β6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAcβ1-3GalβR as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a β1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all β6GlcNAc structures found in mucin-type oligosaccharides.

Original languageEnglish (US)
Pages (from-to)23863-23871
Number of pages9
JournalJournal of Biological Chemistry
Volume266
Issue number35
StatePublished - Dec 1 1991

Fingerprint

Uridine Diphosphate
Biosynthesis
Mucins
Purification
Transferases
Enzymes
Oligosaccharides
Sucrose
Mucin-3
Membranes
Centrifugation
Octoxynol
Electrophoresis
Autoradiography
Sodium Dodecyl Sulfate
Protons
Polyacrylamide Gel Electrophoresis
Epithelium
Nuclear magnetic resonance
Kinetics

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Mucin biosynthesis : Purification and characterization of a mucin β6N-acetylglucosaminyltransferase. / Ropp, P. A.; Little, M. R.; Cheng, Pi-Wan.

In: Journal of Biological Chemistry, Vol. 266, No. 35, 01.12.1991, p. 23863-23871.

Research output: Contribution to journalArticle

@article{95dda0ba094642d8afbe2b89dca81d21,
title = "Mucin biosynthesis: Purification and characterization of a mucin β6N-acetylglucosaminyltransferase",
abstract = "We have purified, to apparent homogeneity, a mucin β6N-acetylglucosaminyltransferase (β6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1{\%} Triton X-100 in the presence of 1 mM Galβ1-3GalNAcαbenzyl (Bzl) to stabilize the β6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Super-flow affinity column equilibrated with 1 mM Galβ1-3GalNAcαBzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3{\%} yield and a specific activity of 70 μmol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the β6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The K(m) values for UDP-GlcNAc and Galβ1-3GalNAcαBzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified β6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAcβ1-3GalβR as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a β1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all β6GlcNAc structures found in mucin-type oligosaccharides.",
author = "Ropp, {P. A.} and Little, {M. R.} and Pi-Wan Cheng",
year = "1991",
month = "12",
day = "1",
language = "English (US)",
volume = "266",
pages = "23863--23871",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "35",

}

TY - JOUR

T1 - Mucin biosynthesis

T2 - Purification and characterization of a mucin β6N-acetylglucosaminyltransferase

AU - Ropp, P. A.

AU - Little, M. R.

AU - Cheng, Pi-Wan

PY - 1991/12/1

Y1 - 1991/12/1

N2 - We have purified, to apparent homogeneity, a mucin β6N-acetylglucosaminyltransferase (β6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Galβ1-3GalNAcαbenzyl (Bzl) to stabilize the β6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Super-flow affinity column equilibrated with 1 mM Galβ1-3GalNAcαBzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 μmol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the β6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The K(m) values for UDP-GlcNAc and Galβ1-3GalNAcαBzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified β6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAcβ1-3GalβR as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a β1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all β6GlcNAc structures found in mucin-type oligosaccharides.

AB - We have purified, to apparent homogeneity, a mucin β6N-acetylglucosaminyltransferase (β6GlcNAc transferase) from bovine tracheal epithelium. Golgi membranes were isolated from a 0.25 M sucrose homogenate of epithelial scrapings by discontinuous sucrose gradient centrifugation. The Golgi membranes were solubilized with 1% Triton X-100 in the presence of 1 mM Galβ1-3GalNAcαbenzyl (Bzl) to stabilize the β6GlcNAc transferase. The solubilized enzyme was bound to a UDP-hexanolamine-Actigel-ALD Super-flow affinity column equilibrated with 1 mM Galβ1-3GalNAcαBzl and 5 mM Mn2+. Elution of the enzyme with 0.5 mM UDP-GlcNAc resulted in a 133,800-fold purification with a 1.3% yield and a specific activity of 70 μmol/min/mg protein. Radioiodination of the purified enzyme followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a single band at 69,000 Da. Kinetic analyses of the β6GlcNAc transferase-catalyzed reaction showed an ordered sequential mechanism in which UDP-GlcNAc binds to the enzyme first and UDP is released last. The K(m) values for UDP-GlcNAc and Galβ1-3GalNAcαBzl were 0.36 and 0.14 mM, respectively. Acceptor competition studies showed that the purified β6GlcNAc transferase can use core 1 and core 3 mucin oligosaccharides as well as GlcNAcβ1-3GalβR as acceptor substrates. Proton NMR analyses of the three products demonstrated that GlcNAc was added in a β1-6 linkage to the penultimate GalNAc or Gal, suggesting that this enzyme is capable of synthesizing all β6GlcNAc structures found in mucin-type oligosaccharides.

UR - http://www.scopus.com/inward/record.url?scp=0026352595&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0026352595&partnerID=8YFLogxK

M3 - Article

VL - 266

SP - 23863

EP - 23871

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 35

ER -